Zuogui and Yougui pills extend lifespan and improve ageing biomarkers via kaempferol-mediated mitophagy in Caenorhabditis elegans

Zuogui pill (ZGP) and Yougui pill (YGP) are classical kidney-tonifying formulas in Traditional Chinese Medicine, widely used clinically but with their potential to delay ageing and improve ageing biomarkers remaining unclear. This study combined network pharmacology and Caenorhabditis elegans models to investigate the anti-ageing effects and mechanisms of ZGP and YGP. Both formulas significantly extended lifespan (ZGP dose-dependently at 5–20 mg/mL; YGP at 20 mg/mL) and improved ageing biomarkers, as evidenced by enhanced motility, reduced lipofuscin accumulation and endogenous ROS levels, and increased resistance to heat and oxidative stress. Network analysis identified quercetin and kaempferol as the top-ranked shared active components. Subsequent experimental validation demonstrated that kaempferol (0.05–0.2 mM) replicated these pro-longevity effects and was shown to act by inducing mitophagy: it triggered an initial decrease followed by a long-term increase in mitochondrial content, concomitant with upregulated expression of mitophagy genes. Crucially, the lifespan-extending effects of kaempferol, ZGP, and YGP were completely abolished in bec-1 and pink-1 null mutants. This study establishes that ZGP and YGP delay ageing and improve ageing biomarkers in C. elegans by activating BEC-1/PINK-1-dependent mitophagy. Kaempferol was identified as a major active component mediating this effect, highlighting a key mechanism for the pro-longevity properties of these traditional formulas.